Cosynthetic factor-1, and its production



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JERRY R.D. Mc CORMICK NANCY H.ARNOLD URSULA HIRSCH PHILLIP A.M|LLER O. SJOLANDER Aug. 15, 1961 J. R. D. MCCORMICK ET AL 2,996,499

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COSYNTHETIC FACTOR-l, AND ITS PRODUCTION Filed June 20, 1958 3 Sheets-Sheet 3 's AL.

IN MILLIMICRONS I I 22012o|30o|34o|58o 42o|4eo 5oo 360 400 44 IN MILLIMICRONS JERRY R.D. McCORMICK NANCY H. ARNOLD URSULA HIRSCH PHILLIP A.MILLER NE ELLO.SJOLANDER ATTORNEY United States Patent 2,996,499 COSYNTHETIC FACTOR-1, AND ITS PRODUCTION Jerry Robert Daniel McCormick, New City, Nancy Hazlett Arnold and Ursula Hirsch, Pearl River, and Philip Andrew Miller, New City, N.Y., and Newell Oscar Sjolander, Saddle River, N.J., assignors to American Cyanamid Company, New York, N. a corporation of Maine Filed June 20, 1958, Ser. No. 743,424 5 Claims. (Cl. 260-2365) This invention relates to a novel substance having biological activity which is elaborated by-various species of the genus Streptomyces. The novel substance, which We have designated as Cosynthetic Factor-1, hereinafter sometimes referred to as CF-l, has the property of stimuating the production of high concentrations of chlortetracycline when added to a fermentation of strains of S. aureofaciens which produce only minimal amounts of chlortetracycline.

In the copending application of John A. Growich and Philip A. Miller, Serial No. 650,821, filed April 5, 1957, there are described and claimed certain new compounds related. to the tetracyclines which have been designated 5a(11a)-dehydrotetracyclines. The 5 a( 1 la) -dehydrotetracyclines are produced by certain strains of S. aureofaciens, such as strain S1308, for example, the morphological and cultural characteristics of which are adequately described in the aforesaid copending application. In addition, viable cultures of S. aureofaciens, strain S1308, as well as several variants thereof have been deposited with the American Type Culture Collection in Washington, D.C. where they have been assigned ATCC Accession Numbers 12748-12751, inclusive. The 5a(11a) -dehydrotetracyclines are largely biologically inactive but they may be converted by a suitable catalytic reduction process to the well-known, broad-spectrum antibiotic tetracycline.

We have now discovered that when the novel substance of this invention is added to a fermentation medium inoculated with S. aureofaciens strain S1308 (ATCC No. 12748), for example, and the culture is grown under standard aerobic conditions, the amount of chlortetracycline produced is increased from about 100-400 micrograms per milliliter to more than 5,000 micrograms per milliliter. In this case, 5 a(11a)-dehydrotetracycline is no longer produced. Just why the addition of Cosynthetic Factor-1 to the fermentation produces such a high concentration of chlortetracycline when that fermentation ordinarily produces only minimal amounts of chlortetracycline is notknown with certainty, and no theory is advanced with respect thereto. It is known, however, that CF-l is not a precursor as is shown by the fact that one microgram of CF-l when added to a sufiicient quantity of S. aureofaciens AT CC No. 12748 results in the biosynthesis of up to 57 milligrams of chlortetracycline beyond that normally produced under the same conditions.

The novel substance of this invention is composed of the elements carbon hydrogen, nitrogen and oxygen. Elementary analytical determinations of purified samples indicate elementary proportions by weight substantially as follows: carbon, 50.33%; hydrogen, 5.00%;

nitrogen, 12.18%; oxygen (direct), 32.58%. The compound is of relatively low molecular weight, 340-360. The empirical formula based on the analytical values above corresponds closely to C 4 15H1qN3O7. The product is soluble in water at pH 6 and is soluble in phenol. It is insoluble in n-butanol, acetone, ether and in water at pH 1-2.

Cosynthetic Factor-1 has an Rf value of 0.07 on a paper chromatogram developed with 1:1 n-butanol-water system and an Rf value of 0.28-0.35 in a 3:1:4 n-

paper and the presence of CF-l is determined by its characteristic yellow-green fluorescence under ultraviolet light.

In a fifty tube Craig countercurrent distribution operation utilizing 1:1 phenol-chloroform as the organic phase and 0.1 N HCl as the aqueous phase, CF-l appears as a single component with a peak concentration at tube 31.

An infrared absorption spectrum of a sample of the compound as obtained from dilute hydrochloric acid solution (acid form) is prepared in a standard manner by mixing with potassium bromide crystals and pressing into a disc. The acid form of the compound exhibits characteristic absorption in the infrared region of the spectrum at the following wave lengths expressed in microns: 2.84, 3.18, 3.35, 3.55, 5.91, 6.02, 6.27, 6.35, 6.49, 6.64, 6.72, 7.02, 7.18, 7.35, 7.49, 7.93, 8.16, 8.36, 8.69, 9.15, 9.30, 9.59, 9.77, 9.90, 10.11, 10.47, 11.40,

11.62, 11.81, 12.56, 12.83, 13.30, 14.48. This infrared;

curve is shown in FIGURE 1 of the accompanying drawings.

The compound as obtained from pH 7 ammonium hydroxide solution (neutral form) when suspended in a potassium bromide pellet exhibits characteristic absorption in the infrared region of the spectrum at the following wave lengths expressed in microns: 3.12, 3.37, 3.55, 6.07, 6.33, 6.63, 7.00, 7.25, 7.44, 7.88, 8.13, 8.35, 8.73,

12.96, 14.50. This infrared curve is shown in FIGURE 2 of the accompanying drawings.

An ultraviolet absorption spectrum determined from a sample of the compound at a concentration of 10.7 micrograms per milliliter in 0.01N HCl, shows characteristic absorption maxima at 230 m 250 mp, 267 my and 377 m corresponding to extinction coefiicient values of 1010, 580, 600 and 693, respectively. This ultra-- violet absorption curve is shown in FIGURE 3 of the accompanying drawings.

An ultraviolet absorption spectrum determined from a sample of the compound at a concentration of 10.7 micrograms per milliliter in 0.01N NH.,OH shows characteristic absorption maxima at 248 III/4., 268mg, 295 mg and 419 mu, corresponding to extinction coefficient values lli...

This curve is These organisms are all species of the genus Streptomyces.

1: S. albus which produces Vitamine B S. rimosus, S.

platensis and S. hygroscopicus, all of which produce oxytetracycline; S. viridiyaciens which produces chlortetracycline and tetracycline; S. aureofaciens which produces puromycin.

The organism that we prefer to use for the production of Cosynthetic factor-1, because of its ability to produce- Patented Aug. 15, 1961 Thus, we have successfully used the following' species of microorganisms to produce Cosynthetic factor larger amounts of this novel substance is a new strain of S. aureofaciens which we have designated as W-S.

The new strain is a member of the species S. aureofaciens since it is a direct descendant of the chlortetracyclineproducing strain of S. aureo'faciens A377 which was isolated from the soil and is describedin the United States patent to' Duggar No. 2,482,055, and the culture is dea general characteristics as do the strains which produce the tetracyclines and differs in the same general manner that the tetracycline-producing and chlortetracycline-producing strains of S. aureofaciens differ from each other,

as has been described in a number of scientific papers which have been published. The data appearing below will serve to illustrate the variation of strain W-S from the original A377 strain available as NRRL 2209.

Streptomyces azireofaciens strain W-S was diiferenti ated from Streptomyces aureofaciens strain A377 (NRRL 2209 by observation of growth characteristics on various media incubated at 26.5 C.

1. Glycerol asparagine beef extract agar Glycerol percent- 1.0 L-aspar in do 0.05 Beef extract dn ()2 KH2P04 -d 0.05 Bacto agar do 1.5 Distilled water, q.s d 100.0 Adjustment with 50% KOH to pH 7.0 Post sterilization pH 7.2

' Streptomyces cureofaciena Strain W- Strain A377 Growth Fair to good, hyallne Good.

- to semi-opaque.

white. Aerial hyphae Sparse to moderate, Slight to fair, white to w e. light gray. Sporulation Sparse, dark gray--." Light gray. Difiusible pigment None Light yellow. Reverse 'White, semi-opaque-.. Yellow to light orangeyellow.

2. Dextrm Czapek-Dox agar Dexh'in percent 1.0 NaNO do 0.2 K HPO do 0.1 M S0.7H2O do 0.05 Kfl do..- 0.0a FeSO4.7H O I do 0.001 Bacto agar do 1.5 Distilled water, q.s do 100.0 Post sterilization pH 7.2

Streptomyces aureofaciens Strain W-5 Strain A377 Growth Very thin, clear hy- Good. aline to translucent Aerial hyphaa, Abundant, mouse gray 1 to lead gray 1 water-white surface r V globules. Sporulation Profuse. Diflusible pigment... Trace, pale yellow. Reverse lear hy Aplgmentous, pink cent white. trace.

4 Color Harmony Manual, third edition, Gontainerborporation of Ame ca AP6 Corn Steep Agar.

4 1 3. AP4 corn steep agar Corn steep rcent 0.4 Sucrose d 1.0 MgSOi.7H O .025 K PO4 0.2 NHQ HPOi 0.2 acto agar 2.0 Tap water, .q. do 103g Post sterilization pH a Streptomgcea aureofaciens' Strain A377 Strain W-5 Growth--. Excellent, white- Excellent. Aerial myce Profuse Abundant, fawn. Sporulatton Profuse, silver gray Profuse, uniform. to beige gray.

Reverse..-

one White to beaver to chocolate.

Light yellow to amber. Light; tan.

Color Harmony Manual, third edition, Container Corporation of Glucose Asparagine Meat Extract Agar.

Wsksmans Agar Potato Slants verse: clear hyaline to white. N o soluble pigments.

Fair growth: clear hyaline to translucent white. None to sparse aerial mycelium: white to beaven Sparse sporulation. Reverse: colorless to white. N0 soluble pigment.

Excellent growth:

white. Aerial mycelium: moderate to abundant, silver gray. Moderate to abundant sporulation. Reverse: white to bcaven N o soluble pigment.

Good growth; white to light yellow. Aerial myceliurn: fair to good, white becoming beaver 1 to ashes. Fair sporulation. Reverse: light yellow 1 to rose taupe to taupe brown. Yellow to amber soluble pigment.

Excellent, smooth,

moist nodulated growth: light fawn 1 to copper tan 1 to brick red. No aerial mycelium. Light fawn 1 soluble pigment.

Slight but; definite white growth collar, little significant pH change. No apparent peptonization in 14 days.

Good growth. Aerial hyphae white becoming increasingly gray with increase with spore formation. Reverse: light yellow to pink'orange. Trace: yellow-orange soluble pigment.

Excellent growth.

Profuse aerial mycelium. Profuse sporulation: fawn. Reverse: tan. Light amber soluble pigment.

Good growth. Aerial hyphae fair becoming abundant: white to taupe brown. Reverse: camel 1 to adobe brown. Light yellow soluble pigment.

Profuse, moist,

smooth, nodulated growth: light brown yellow 1 to beige to cedar. No soluble pigment.

Slight white to pale yellow growth collar. Little significant pH change nor apparent peptonization in 14 days.

America.

1 A196 Agar:

Sucr

Percent Corn Steep- Bacto (refined) agar 5. Microscopic observations Mycelial and spore morphology of Streptomyces aureofaciens strain W-S is apparently similar to that of the original strain A377.

Viable cultures of S. aureofaciens strain W-5 which produce the novel substance of this invention have been deposited with the American Type Culture Collection in Washington, DC. where this strain has been assigned ATCC Accession Number 13,190.

The conditions for fermentation with the new strain of S. aureofaciens of this invention are generally the same as those presently used for cultivating other S. aureofaciens strains. That is, the fermentation medium contains the usual nutrients and essential mineral elements. Suitable substances which may provide the necessary nutrients include starch, dextn'n, cane sugar, glucose, molasses, soybean meal, peanut meal, yeast, meat extracts, peptone, ammonium sulfate, urea, corn steep liquor, distillers solubles, fish meal and other conventional substances. The inorganic salts that may be used include calcium carbonate, ammonium sulfate, ammonium chloride, and the various trace elements such as manganese, cobalt, zinc, copper, iron and the like.

As distinguished from fermentations of 5a(11a)-dehydrotetracycline producing culture S1308 and its variants which produce small quantities of chlortetracycline, we have not observed any tetracycline or chlortetracycline production when the new S. aureofaciens strain W-S is grown under standard aerobic conditions.

The CF-l producing culture, suchas the new strain described above, is grown aerobically in a suitable inoculum medium. The inoculum. is then transferred and grown in a suitable fermentation medium and the fermentation is carried out at a temperature of about 22 C. to 32 C. for from 48 to 168 hours on a rotary shaker. During the fermentation the pH is usually maintained between about 5.5 and 7.5. After the fermentation is complete, the pH of the mash usually ranges from about 6-7. The mash is then filtered without a pH adjustment. The CF-l may then be extracted, isolated and purified in any suitable manner.

A preferred extraction process involves filtering the mash at the prevailing pH (6-7). The pH of. the filtrate is then adjusted to pH 8-9 with ammonium hydroxide and saturated with ammonium sulfate. A suitable carrier such as Arquad 16, which is a long chain alkyltrimethylammonium chloride in which the alkyl groups consist of 90% hexadecyl, 6% octadecyl and 4% octadecenyl, is added and the mixture is then extracted with a lower alkanol, i.e., n-butanol. The n-butanol extract is then. adjusted to pH 1.5-2.0 with concentrated hydrochloric acid and is back-extracted with water and the two phases are separated. The aqueous phase which contains the CF-l is then concentrated under reduced pressure. The

atomaceous earth column in the usual manner the column being developed with a buffered. solution of nbutanol. The CF-l rich cuts are then extracted into water and the back-water extract is concentrated under vacuum, the aqueous extract is then chromatographed on,

a Florisil column. Florisil is an activated magnesium silicate of approximately the following composition:

MgO15.5% :o.s%; SiO -84.0% i0.5%

and Na SO -0.5%. The CF-l is eluted from the Florisil column with methyl alcohol containing a small amount of water. The CF-l rich cuts are concentrated,

adjusted to pH 1 with hydrochloric acid and filtered.

The filtrate is cooled and seeded. The resulting crystals of CF-l are collected by filtration, washed and Vacuum dried. The crude product may be purified in a standard manner by recrystallization from 0.1 H01.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 lnoculum preparation A typical medium used to grow the primary inoculum is prepared according to the following formula:

Sucrose grams 30.0

Calcium carbonate do 7.0

Water to 1000 milliliters.

Eight milliliter aliquots of this medium are placed in each of a series of 8 inch test tubes and sterilized by autoclaving for 20 minutes under 15 pounds per square suspension is used to inoculate each of the tubes con taining an 8-milliliter portion of the inoculum medium shown above. The inoculated shaker tube is then incubated for 24 hours at 28 C. on a reciprocating shaker operating at 116 oscillations per minute.

EXAMPLE 2 Fermentation I A fermentation medium is prepared according to the following formula:

(NH.;) grams 5.0 CaCO do 9.0 NH Cl do 1.5 MgCl .6H O do 2.0

aqueous concentrate is then chromatographed on a di- (5 FeSO .7H O do 0.06

Cornstarch do 55.0

Water to 1000 milliliters.

Twenty-five milliliter portions of the medium are placed in 250 milliliter Erlenmeyer flasks, and 0.5 milliliter of lard oil is added to each flask. The flasks containing the fermentation medium and lard oil are sterilized'in an autoclave for 20 minutes under 15 pounds per square.

inch pressure. Following sterilization and cooling, 1 milliliter of the inoculum, prepared as in Example 1, is added to each flask and the fermentation is carried out at 25 C., for 120 hours, on a rotary shaker operating at 180 revolutions per minute. The mash is assayed and found to contain one microgram per milliliter of Cosynthetic Factor-1.

- EXAMPLE 3- Extracled from whole mash A 200 liter portion of fermentation mash prepared in a pilot tank fermentor with CF1 producing strain of S. aureofaciens W-S grown in the medium described in Example 2 and assaying one microgram of CF-1 per milliliter is filtered at pH 6-7 using Hyflo Super-Col (diatomaceous earth) in an amount approximating 15% of mash volume. The filter cake is washed withsuflicient water to give a total combined neutral filtrate volume equal to the starting mash volume. The CF-1 potency of the combined neutral filtrate is about one microgram per milliliter. The combined neutral filtrate is concenhated to one-quarter starting mash volume under vacuum at 60 C. Thus the final volume of concentrated combined neutral filtrate is 50 liters.

The combined neutral filtrate concentrate is adjusted to pH 8-9 with concentrated ammonium hydroxide and then saturated with ammonium sulfate. Arquad 16 (cetyltrimethylammonium chloride) is added to this solution in the proportion of 10 milliliters of a 50% Arquad 16 solution in isopropanol to each liter of combined neutral filtrate concentrate, and the mixture stirred for onehalf hour. The mixture is then combined with an equal volume of n-butanol and stirred for another one-half hour. The resultant mixture is centrifuged in order to separate the phases. This extraction is repeated using 10 milliliters of 50% Arquad 16 per liter of combined neutral filtrate concentrate and another equal volume of n-butanol. The n-butanol extracts are then combined to yield a final volume of 100 liters. The combined nbutanol extracts are concentrated to one-quarter starting volume under vacuum at 60 C. Thus the final volume of the concentrated combined n-butanol extracts is 25 liters.

The 25 liters of concentrated, combined u-butanol extracts are adjusted to pH 1.5-2.0 with concentrated hydrochloric acid. A S-liter quantity of water and 200 liters of methylene chloride are added and the mixture stirred well for one-half hour. The S-liter aqueous layer is separated from the organic phase. The pH of the -liter volume of back water extract is adjusted .to 7.0 with ammonium hydroxide solution before it is concentrated under vacuum at 60 C. to 5-1.0% of the original fermentationmash volume. One liter of back water extract concentrate is thereby obtained.

Isolation per milliliter of concentrate; 4 A Celite 545 column is prepared in the following manner: A phosphate butter solution is made with 10.75 grams of K HPO per liter of, water andadjusted to pH 8.0 with H PO Celite 545 is mixed thoroughly with pH 8.0 butler solution in the '-proportions of 2 grams of Celite 545 per milliliter of buffer solution. A 9-inch diameter column is packed to a height of 2 feet with this buttered Celite 545. The

Celite 545 containing the solution of crude Cosynthetic' Factor-1 (concentrate of back water extract) is packed on top of the bufiered Celite 545 column. The column is developed with n-butanol buffered to pH 8.0 as above. The combined CF-l rich cuts are mixed with an equal volume of water and two volumes of methylene chloride. The mixture is shaken in a separatory funnel. The organic phase is reextracted with another one-half volume of water. The total volume of combined back water extract amounts to 8 liters which is concentrated under vacuum at 40 C. to 200 milliliters.

Purification -A Florisil column is prepared in the following manner: Florisil is packed into a 3-inch diameter column to a height of 18 inches. The column is washed with 0.01N NH OH (aqueous) then with methyl alcohol containing 10% water, and finally with 0.01N NH OH (aqueous) again. The approximately 200 milliliters of combined back water extract concentrate is placed on top of the packed and washed Florisil column and allowed to pass through; followed by a one liter volume of 0.01N NH OH (aqueous) and 500 milliliters of water. Cosynthetic Factor-1 is then eluted from this column by means of methyl alcohol containing 10% water. The combined CF-l rich cuts are adjusted to pH 7.0 with carbon dioxide, then concentrated at less than 40 C. and under vacuum to yield approximately 100 milliliters of an aqueous solution of CF-l.

The combined CF-l rich concentrate is adjusted to pH 1.0 with hydrochloric acid, heated to C. and filtered through Whatman No. 4 filter paper. The filtrate is cooled, seeded, and allowed to stand overnight at 15 C. The resulting crystals are collected by filtration, washed first with 0.1 N hydrochloric acid solution, then with water, and finally with acetone, followed by ether, and vacuum-dried at 40 C. overnight to obtain 92 milligrams of crude. product.

The crude crystals are dissolved in 0.1N hydrochloric acid solution, heated to 90 C., and cooled in ice-water at about 5 C. to promote crystallization. The newlyformed crystals are first washed with cold (10 C.) 0.1 N hydrochloric acid solution, then with cold water, and dried under vacuum at less than 40 C. for 3 hours to obtain 62 milligrams of the acid form of Cosynthetic Factor-1, melting with decomposition at 280-285 C. The neutral form of the product is obtained by dissolving 5 milligrams of the acid form of CF-l in 3 milliliters of hot water by the addition of ammonium hydroxide to pH 7.5. The crystalline neutral form precipitates from solution on cooling. The crystals are washed with cold water and then vacuum dried at 40 C. The chemical analysis of CF-l and its other chemical, physical, and biological properties have already been described.

EXAMPLE 4 25 C., and assayed for the amount of chlortetracycline A 120-hour fermentation with S. aureofaciens W-S is carried out following the procedure of Example 2. The resulting mash is filtered without pH adjustment to obtain a W- filtrate.

An S. aureofaciens S1308 fermentation is prepared and carried out as in Example 4 but substituting 0.08 milliliter of this W-S neutral filtrate for the crystalline CF-l used in Example 4. The harvest mash assays are as follows:

Miililiters of W-5 Neutral Filtrate per milliliter of Chlortetraeycline S1308 mash Produced (Turbidimetric Assay) By comparison of these results with those on pure CF-l in Example 4 it will be-seen that the W-5 mash contains 0.85 micrograms of CF-l per milliliter.

EXAMPLE 6 All of the strains of Streptomyces listed below are grown in separate primary fermentations, and after growth, aliquots of the harvest mashes are added to Erlenmeyer flasks containing the medium shown in Example 2 so that the final total volume of primary fermentation mash plus medium in each flask is 25 milliliters. The flasks and their contents are then sterilized by autoclaving for 20 minutes under 15 pounds per square inch pressure, and subsequently cooled to 25i5 C.

- An inoculum of S. aureofaciens S1308 (ATCC 12748) is prepared according to the techniquegiven in Example 1. At the termination of the 24-hour incubation period, flasks containing the 25 milliliters of combined fermentation mash prepared above are each inoculated with 1.0 milliliter of the S. aureofaciens S1308 inoculum. There inoculated flasks are incubated at 25 C. for 120 hours on a rotary shaker operating at 180 revolutions per minute. At the end of the fermentation period, each flask is assayed fluorometrically for chlortetracycline and the CF-1 content of each primary fermentation mash is calculated with the results obtained being shown below:

EXAMPLE 7 An S. aureofaciens W-S neutral filtrate is prepared as described in Example 5. An S. aureofaciens E504 (ATCC 13,191) fermentation is prepared and carried out as in Example 4, but substituting different volumes;

of the S. aureofaciens W-5 neutral filtrate for the crystalline CF-l used therein. The harvest mash assays are as follows:

Spectrophotometric Assay, meg/ml.

'Iligrbidimetri/c:

say, meg.

The addition of Cosynthetic Factor-1 to the above S.

aureojaciens fermentation results in the increased production of 7-chloro-6-demethyltetracycline.

EXAMPLE 8 tion of 0.10 milligrams of DMTD per milliliter of fermentation medium. All six flasks are then sterilized and cooled. An S. aureofaciens S1308 fermentation is prepared and carried out as in Example 4 but substituting 0.08 milliliter of the S. aureofaciens W-S neutral filtrate for the crystalline CF-l used therein. The harvest mash assays are as follows:

Ohlortetrn- Tetracycline Milliliters of W-fi Neutral Fll- DMTD, cy ine Spectrotrate per Miliiliter of S1308 mg/ml. Fluorometric photometric Mash Assay, Assay,

meg/ml. meg/ml.

Carbon 50.33 Hydrogen 5.00 Nitrogen 12.18 Oxygen 32.42

said substance having a molecular weight of 340360, said substance having a melting point of 280-285 C., said substance when dissolved in 0.01N HCl exhibits characteristic ultraviolet absorption maxima at 230 m 250 my, 267 m and 377 my, corresponding to extinction coeflicient values lia) of 1010, 580, 600 and 693, respectively, and when dissolved in 0.01N NI-I OH exhibits characteristic ultraviolet absorption maxima at 248 m 268 m 295 my. and 419 m corresponding to extinction coeflicient values of 972, 684, 349 and 1215, respectively, and when suspended in a potassium bromide pellet the acid form of said substance'exhibits characteristic absorption'inthe infrared region of the spectrum at the following wave lengths expressed in microns: 2.84,3;18, 3.35, 355,591, 6.02, 6.27, 6.35, 6.49, 6.64, 6.72, 7.02, 7.18, 7.35,"7;49, 7.93, 8.16, 8.36, 8.69, 9.15, 9.30, 9.59, 9.77, 9.90, 10.11, 10.47, 11.40, 11.62, 11.81, 12.56, 12.83, 13.30, 14.48, and when suspended in a potassium bromide pellet the neutral form of said substance exhibits characteristic absorption in the infrarcd'region of the spectrum at the following wave lengths expressed in microns: 3.12, 3.37, 3.55, 6.07, 6.33, 6.63, 7.00, 7.25, 7.44, 7.88, 8.13, 8.35, 8.73, 9.25, 9.55, 9.63, 9.84, 10.28, 10.42,11.57,,1115,1260,12,96, 14.50. 7 f 2. A process for the production or cosynrtrefiefactorq which comprises cultivating a cosynthe'tic factor-'il'prbducing strain selected from the group consisting of Streptbmyces'aureofaciens, Streptamyces albus, Streptomyces rimosus, Streptomyces platensis, Streptomyces 'viridifa ciens, Streptomyces hygroscopiczis,'Streptomyces griseus and Streptomyces albo-niger in an aqueous nutrient mec'li'um containing assirniable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic conditions until substantial cosynthetic factor activity is imparted to said medium, and recovering the cosynethie factor so-produced, said cosynthetic factor being the substance as described in claim 1. V V

3. A process for the production of cosynthetic factor-1 which comprises cultivating Streptomyces aureofaciens ATCC 13,190 in an aqueous nutrient medium containing assiinilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic conditions until substantial cosynthetic factor activity is imparted'to said medium, and recovering the cosynthetic factor so-produced, said cosynthetic factor being the substance as described in claim 1. a

4. A process for the production of cosynthetic factor-1 which comprises cultivating a cosynthetic factor-l'producing strain selected from the group consisting of Streptomyces aureofaciens, Streptomyces albus, Streptpmyces. rimosus, Streptomyces platensis, .Streptomyccs .viridifa ciens, Streptomyces hygroscopicus; :Streptomyces griseus and Szreptomyces albo-niger in *an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts under submerged aerobic conditions for a period of from about 48 to 168'hours and at a temperature of from about 22 to 32 C., and recovering the cosynthetic factor so-produced, said cosynthetic factor being the substance as described in claim 1.

r .A processfor the production of cosyntheticiactor-f which comprises cultivating Streptomyces aureofaciens' ATCC 13,190 an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic conditions for a References Cited in the file of this patent UNITED STATES PATENTS 2,482,055 Duggar Sept. 13, 1949 2,516,080 Sobin et a1,' July 18, 1950 2,712,517 Gourevitch .et a1. July 5, 1955 2,734,018 Minieri Feb. 7, 1956 2,763,591 Hatch .Sept. 18, .1956 2,805,185 Hermann et a1 Sept. 3, 1957 2,847,471 Vandeputte Aug. 12, 1958 2,878,289 McCormick et a1 Mar. 17, 1959 2,923,668 Goodman Feb. 2, 1960 2,931,798 Umezawa et a1. Apr. 5, 1960 FOREIGN PATENTS 713,795 Great Britain Aug. 18, 1954 744,965 Great Britain Feb. 15, 1956 781,843 Great Britain Aug. 28, 1957 OTHER REFERENCES. 1

Sekizawa: Jour. of Biochemistry, vol. 42, No. 2, 1955, pp. 217-219.

' Doerschuk et al.: Jour. Am. Chem. Soc., Sept. '5, 1955, 13.4687. r

Minieri et al.: Science, vol. 122. p. 1234.

Stevens et a1.,*] our. Am. Chem. Soc, vol. 78, pp. 1515- 1516, Apr. 5, 1956.

Sel'zer et a1., Antibiotics and Chemotherapy, voL'VlI, No. 6,'pp. 292-296. r

. Smith et al;, Iour. OrgJChen'r, May 1958, pp. 721-724, vo1.23. "'5' s is Boothe et al., I our. Am. Chem. Soc., Apr; 5, 1958, vol. 80, pp. 1654-1657. s 1 

1. A COSYNTHETIC FACTOR, SAID SUBSTANCE BEING SOLUBLE IN PHENOL AND IN WATER AT PH>6, INSOLUBLE IN BUTANOL, ACETONE, ETHER AND WATER AT PH 1-2, SAID SUBSTANCE CONTAINING THE ELEMENTS CARBON, HYDROGEN, NITROGEN AND OXYGEN AND HAVING AN EMPIRICAL FORMULA CORRESPONDING TO THE FOLLOWING ANALYSIS:
 5. A PROCESS FOR THE PRODUCTION OF COSYNTHETIC FACTOR-1 WHICH COMPRISES CULTIVATING STREPTOMYCES AUREOFACIENS ATCC 13,190 IN AN AQUEOUS NUTRIENT MEDIUM CONTAINING ASSIMILABLE SOURCES OF CARBOHYDRATE, NITROGEN AND INORGANIC SALTS UNDER SUBMERGED AEROBIC CONDITIONS FOR A PERIOD OF FROM ABOUT 48 TO 168 HOURS AND AT A TEMPERATURE OF FROM ABOUT 22 TO 32*C., AND RECOVERING THE COSYNTHETIC FACTOR SO-PRODUCED, SAID COSYNTHETIC FACTOR BEING THE SUBSTANCE AS DESCRIBED IN CLAIM
 1. 